Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1994-12-29
1997-05-20
Ketter, James
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
536 245, C12P 1934, C07H 2100
Patent
active
056311150
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a polyribonucleotide having a thermodynamically stable loop structure and ribozyme activity, a DNA that codes for said polyribonucleotide, an expression vector containing said DNA, a host cell transfected with said expression vector, and a process for cleaving a specific site of a substrate polyribonucleotide by said polyribonucleotide having ribozyme activity.
BACKGROUND ART
The (+) chain of satellite RNA of tobacco ringspot virus and the (+) chain and (-) chain of avocado sunblotch viroid are cleaved by their own catalytic activity in the presence of Mg.sup.2+ (Science 231, 1577-1580 (1986)). The RNA structure necessary for this cleavage activity has been determined and named as Hammerhead ribozyme (Nucleic Acids Res. 14, 3627-3640 (1986)). The nucleotide sequences in the vicinity of the cleavage sites of these RNA possesses common sequences, and the secondary structure of these RNA was predicted from this common sequences. Uhlenbeck designed a short chain RNA fragment of 19 mer based on these common sequences, and indicated that said fragment catalytically cleaves RNA of 24 mer (Nature, 328, 596-600 (1987)).
In addition, besides viroid and vital satellite RNA, the transcript of newt satellite DNA is also reported to have ribozyme nucleotide sequences (Cell, 48, 535-543, (1987)).
The inventors of the present invention chemically synthesized two types of 21 mer RNA having nucleotide sequences in the vicinity of the cleavage site of this newt satellite DNA transcript. When one of the RNA was added to the other, a cleavage reaction was found to occur at the same site as that in nature (FEBS Lett., 228, 228-230, (1988)). In addition, based on this result, the inventors of the present invention found a process for cleaving other RNA or polyribonucleotide molecules using ribozyme (Nucleic Acids Res., 17, 7059-7071, (1989)).
On the other hand, a cleavage reaction was also caused on the (-) chain of the satellite RNA of tobacco ringspot virus, and that cleavage has been determined to occur at a specific site (Nature 323, 349-353 (1986)). In addition, the minimum region of RNA necessary for this cleavage has also been recently clarified (Biochemistry, 28, 4929-4933 (1989)). RNA having this catalytic activity is composed of 50 nucleotides, and a model having a hairpin loop structure within this RNA has been advocated. This RNA has been given the name hairpin ribozyme. The present inventors and other research groups converted the nucleotides of this hairpin ribozyme to other nucleotides, and used those results to identify several nucleotides that are important in the cleavage reaction (Nature 354, 320-322 (1991), Nucleic Acids Res. 19, 6833-6838 (1991)). Further, the group of Burke et al. has recently identified the base sequence which is important in the cleavage reaction and ligation reaction of hairpin ribozyme by in vitro selection method using DNA having random variation and PCR (polymerase chain reaction) (Gene & Development 6, 129-134 (1992)). The present inventors have determined that catalytic reaction proceeds even for RNA deleting the hairpin loop portion (Nucleic Acids Res. 19, 6833-6838 (1991)).
In addition, progress in RNA synthesis techniques in recent years has made it possible to obtain RNA in large volume, and thus, researches on higher-order structures of RNA and its physicochemical properties are increasingly made. Bacteriophage T.sub.4 mRNA and E. coli 16S ribosomal RNA contain 5'CUUCGG3' sequences in high frequency, and it has been determined that the hairpin loop structure formed by this sequence is thermodynamically stable (Pro. Natl. Acad. Sci. USA, 85, 1364-1368, (1988): Nature, 346, 680-682, (1990)).
The present inventors prepared a polyribonucleotide wherein the nucleotide sequence of a hairpin loop which exists at one location within the ribozyme is the above-mentioned thermodynamically stable 5'CUUCGG3' sequence, and they found that this polyribonucleotide possesses high ribozyme cleavage activity and accomplished the present inventi
REFERENCES:
Barinaga Ribozymes: Killing the messenger Science 262 1512-1514 1993.
Stull et al. Antigene, ribozyme and aptamer nucleic acid drugs: progress and prospects PharmaceuticaI Res. 12 465-483 1995.
Gerry A. Prody, et al., "Autolytic Processing of Dimeric Plant Virus Satellite RNA", Science, 231, 1577-1580 (1986).
Cheryl J. Hutchins et al., "Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid", Nucleic Acids Research, 14, 3627-3640 (1986).
Olke C. Uhlenbeck, "A small catalytic oligoribonecleotide", Nature, 328, 596-600 (1987).
Lloyd M. Epstein et al., "Self-Cleaving Transcripts of Satellite DNA from the Newt", Cell, 48, 535-543 (1987).
Makoto Koizumi et al., "Construction of a series of several self-cleaving RNA duplexes using synthetic 21-mers", FEBS Letters, 228, 228-230 (1988).
Makoto Koizumi et al., "Design of RNA enzymes distinguishing a single base mutation in RNA", Nucleic Acids Research, 17, 7059-7071 (1989).
Jamal M. Buzayan et al., "Non-enzymatic cleavage and ligation of RNAs complementary to a plant virus satellite RNA", Nature, 323, 349-353, (1986).
Arnold Hampel et al., "RNA Catalytic properties of the Minimum (-) sTRSV Sequence", Biochemistry, 28, 4929-4933 (1989).
Akira Sekiguchi et al., "Mutagenesis and self-ligation of the self-cleavage domain of the satellite RNA minus strand of tobacco ringspot virus and its binding to polyamines", Nucleic Acids Research, 19, 6833-6838 (1991).
George Slim et al, "Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes", Nucleic Acids Research, 19, 1183-1188 (1991).
Craig Tuerk et al, "CUUCGG Hairpins: Extraordinarily Stable RNA Structures Associated With Various Biochemical Processes", Proc. Natl. Acad. Sci. USA, 85, 1364-1368, Mar. 1988.
Chaejoon Cheong et al, "Solution Structure of an Unusually Stable RNA Hairpin, 5'GGAC(UUCG)GUCC", Nature, 346, 680-682 (1990).
Burke et al., "Genes & Development", vol. 6, (1992), pp. 129-134.
John M. Burke et al, "Novel guanosine requirement for catalysis by the hairpin ribozyme", Nature, vol. 354, Nov. 28, 1991, pp. 320-322.
Ignacio Tinoco et al, "Solution structure of an unusually stable RNA hairpin 5'GGAC(UUCG)GUCC", Nature, vol. 346, Aug. 16, 1990, pp. 680-682.
Sekiguchi et al, "Mutagenesis and self-ligation of the self-cleavage domain of the satellite RNA minus strand of tobacco ringspot virus and its binding to polamines", Nucleic Acids Res., vol. 19, (1991) pp. 6833-6838.
Ohtsuka et al, "Ribozymes designed to inhibit transformation of NIH3T3 cells by activated c-Ha-ras gene", GENE (Amsterdam), vol. 117, No. 2, (1992), pp. 179-184.
Koizumi Makoto
Ohtsuka Eiko
Brusca John S.
Ketter James
Sankyo Company Limited
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